Isolated population of plant single cells and method of preparing same

ABSTRACT

This invention is a method of minimizing the variation of cell growth and production through homogeneous cell line development. To be more specific, it is the method of isolating and proliferating single cell clone from cambium to promote the stability of the plant-derived biologically active substances production by solving the problems of decrease in cell growth and the productivity during the long term culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/063,929 filed on Feb. 15, 2008, which is a U.S. national phase ofInternational Application No. PCT/KR2006/001544 filed on Apr. 26, 2006,which claims the benefit of Korean Application No. 10-2005-0103445 filedon Oct. 31, 2005, the contents of which are hereby incorporated byreference in their entirety.

TECHNICAL FIELD

Plant has been used very importantly not only as our food supply butalso as the source of extensive chemical substances including,pharmaceuticals, fragrances, colors, agricultural chemicals and dyesetc. Biologically active compounds that are produced from plants aremostly secondary metabolites. There is a greater interest on thesecondary metabolites, such as alkaloid, allergen, amino acid,anthraquinone, antileukaemic agent, antimicrobial agent, antitumoragent, antiviral agent, enzyme, flavonoids, insecticide, opiate,perfume, pigment, vitamin, and polysaccride etc., because most of themwork as physiologically active substances. According to Zhong (2002),there are about 100,000 known plant secondary metabolites and more than25% of the medicine that are practically used is plant-derivedsubstances. Every year, novel secondary metabolites are discoveredcontinually.

In the method of obtaining these metabolites, there are many problemssuch as difficult chemical synthesis in spite of the recent astonishingdevelopments of the organic chemistry, demolition of the nature due toexploitation and environmental pollution and changes of the content ofmetabolites and increase of the production cost depending on the cultureconditions, e.g., season, region and climate. Therefore, there are ongoing active attempts to produce secondary metabolites through in vitroculture technique which has advantages of controlling the adequateexternal environmental conditions and producing on a large scale even ina small space.

BACKGROUND ART

According to Korean Patent No. 0130100, production of biologicallyactive substances through plant cell culture has more advantages thandirect extraction from the plant. Plant cell culture is considered as anoptimal method for continual production which is not influenced byenvironment and for solving the pending problems like destruction ofecology. Nail & Roberts (2004), however, indicated slow growth rate andlow productivity of the plant cell culture for the secondary metaboliteproduction. To solve this problem, there have been studies of theoptimization of the media, culture conditions, process and elicitationfor higher productivity etc. (Zhong 2002). As disclosed in WO93/17121,various media was used to culture diverse Taxus for the increase in cellgrowth rate and paclitaxel productivity. Based on the results of theexperiments, elicitation conditions for paclitaxel mass production wasindicated. Despite the improvements to the production of valuablesecondary metabolites, variability is still a major issue for theproduction of paclitaxel from Taxus and other valuable substances fromnumerous plant systems.

Production of secondary metabolites through large scale plant cellculture is commercially possible only when there is a stable maintenanceof rapid cell growth and high metabolite production during long termculture. The ability of the cell lines that could produce distinctmetabolites are not stable which cause the cell lines to lose theirinitial productivity through subcultures; it is not too much to say thatsuccess and failure are depended on how we overcome these problems.

In plant cell culture, although the cells are derived from one plant,metabolite productivity of each cell line is different and unstable.Therefore, establishing the cell lines that have high productivity andgenetic stability is most important than anything else.

Cell Lines Derived from Single Cells & and Multiple Cells

Plant cell lines derived from single cells have lower variability thanthe cell lines derived from multiple cells; this results in higherproductivity. In preceding inventions, stem, root, seed, needle and leafwere used as the best explants for cell line induction. These stem,root, seed, needle and leaf are tissues that are composed of the cellswith distinct functions and morphology. Callus, cell lines derived fromthese tissues is not of one kind. Therefore, there are limitations onthe attempts to reduce the productivity variation of the callus derivedfrom the tissues consisted of multiple cells.

Cell Aggregation

One of the distinguishing characteristics of plant cell culture is cellaggregation. According to Korean Patent No. 0364478, diameter of theplant cell is 30-300 μm which is about 30 times bigger than the animalcell. Because plant cell walls have natural tendency to adhere together,it is not possible to obtain suspension which consists only of dispersedsingle cells. The proportion and the size of cell aggregates varyaccording to plant variety and the medium in which the culture is grown.Nail & Roberts indicated that cell aggregation leads to a difference inlocal environment between interior and exterior of the cells, which canresult in culture heterogeneity and ultimately leads to changes ingrowth and metabolism.

The purpose of suspension culture is to obtain pure single cells. Toaccomplish this objective, filtration, maceration and protoplast cultureby using enzyme were used. However, filtration and maceration do notprovide complete pure single cells. Protoplast culture technique whicheliminates the cell wall is the most reliable method for generatingsingle cells, but the enzyme used for the protoplast culture cause cellwall damages or breakages that result in the change of cell physiology.Moreover, hydrophobic secondary metabolites such as paclitaxel can bestored in the cell wall, so the changes in the cell wall have profoundrelationship with productivity.

Also, cell aggregation has long been a major obstacle to the accuratemeasurement of cell growth by number and to biochemical assays toindividual cells. According to Nail & Roberts (2004), if single cellculture is possible, it will readily provide faster information aboutthe behavior of cell units in the culture such as biosynthesis, storage,and degradation etc. of secondary metabolites.

Dedifferentiation

The dedifferentiated cell line, which is callus, shows great variabilityin the production of secondary metabolites due to somaclonal variation.Callus derived from the permanent tissues such as leaves, stem, root andseed that are composed of the cells with distinct functions andmorphology usually show dramatical changes even on slightly differentmicro-environments because it is a secondary meristem formed bydedifferentiation. Due to this sensitivity, Hirasuna et al. (1996)investigated to identify the cell culture conditions, especially initialcell density, subculture interval and temperature, and to maintain themas precisely as possible.

Scale Up

In order to produce secondary metabolites through plant cell culture forcommercialization, scale up is essential. Bioreactor has been appliedfor mass production after many patents and articles were published,reporting about successful production of metabolites through cellculture in a laboratory scale. According to Korean Patent No. 0290004,application of bioreactor for mass production provides very differentculture environment from the flask in a laboratory scale which resultsin the decrease in growth rate and productivity and change in themetabolites. When the bioreactor is applied for mass production, changesin growth rate, productivity and metabolites have become problems incommercialization of biologically active substances through cellculture. In the scale up of plant cell cultures, a bioreactor whichreceives the air through exterior power and/or a bioreactor withimpeller by considering the efficiency of the mixing and aeration arepreferred. However, cell viability decreases abruptly in the bioreactorbecause plant cells are weak for shear. Therefore, a method to reduceshear is necessary. The cause of the shear sensitivity of the plant cellis explained by its large size, rigid cell wall, aggregation andextensive vacuolate (Yokoi, et al., 1993). To solve these problemsassociated with the bioreactor, a low shear generating bioreactor wasinvestigated in the past by controlling its agitating speed andmodifying the impeller type. However, it still bears negative resultsbecause the cell lines could not overcome the differences of themicroenvironment.

Cryopreservation

Cryopreservation allows the long term cell maintenance by ceasing mostof the metabolism of the cells in the extremely low temperature. Itsignifies the recovery of the cells without genetic, characteristics andbiosynthetic variation after cryopreservation. By usingcryopreservation, loss of the cells from contaminations could beeliminated and the genetic variation in the continuous cell lines couldbe minimized. In cGMP, the preservation of the cell lines for a longperiod is mandatory for the stable supply of raw materials. Usually,cultured animal cells could undergo cryopreservation for many years, butthe similar cryopreservation technique is much more challenging forcultured plant cells. Cultured plant cells are heterogeneous and showdiversity in physiology and morphology. Therefore, plant suspensioncells require many processes for cryopreservation and inadequatecryopreservation could cause variability.

Conditioning Factors

Kim et al. (2000) demonstrated that cell division can be stimulated ifsome media from actively dividing cultures was added to the culturesthat lost cell division ability. In the production of anthocyaninthrough rose suspension culture, the productivity increased when somemedia of strawberry suspension culture was added to the rose suspensionculture. In this way, the substances that were produced and secretedfrom the cultured cells to stimulate the cell growth or the productionof the secondary metabolites are called conditioning factors. Yet, theseconditioning factors have not been identified concretely and there areonly some understanding of conditioning factors acting as chemicalsignals for the cell growth and metabolite production. Also, there arefew reports on the potent substances, such as phosphates and calmodiumwhich could be considered as conditioning factors. Conditioning factorscan be supplied through conditioned media or helper cells.

Perfusion Cultivation

Among the cell culture methods, there is a batch cultivation involvingthe inoculation of the cell and the media together in the beginning andno further nutrition supplementation. Also, there is a continuouscultivation, involving the supplementation of the new media as the spentmedia that contains metabolites is retrieved simultaneously at aconsistent speed during the culture period for the prevention ofnutrition depletion.

Batch cultivation is difficult in the commercial level due to its lowproductivity. Among the continuous culture methods, perfusioncultivation is receiving much attention these days. In perfusionculture, the cells are remained in the bioreactor, and new media issupplied as the spent media that contains metabolites is retrieved.

According to Zhang et al. (2000), elicitation is one of the mosteffective ways to promote the secondary metabolites production in cellculture. Elicitation encourages secondary metabolite synthesis, but itinduces cell growth inhibition and the rapid decrease in the cellviability. Hence, secondary metabolite synthesis by elicitation could bemaintained only for a short period and it is very limited. As Wang etal. (2001) presented, perfusion cultivation is a strategy to minimizethese negative effects by elicitation and to maximize the productivity.

Wang et al. (2001) and Wu & Lin (2003) reported as follows. Secondarymetabolites that are produced by elicitation are stored inside the cell(vacuole or cell wall) or released outside the cell (media). During theprocess of culture, releasing secondary metabolites from the cell andremoving it from the media could bring easier purification and coulddiminish the feedback inhibition of biosynthesis and degradation andconversion of the products. Therefore, by retrieving the spent media andsupplying with a new media, secretion of internal and externalmetabolites could extend the viability and biosynthesis of the cells,which could remarkably increase the productivity.

Storage and the secretion of secondary metabolites showed greatdifferences depending on the cell lines. Taxus media cell line(Wickremesinhe and Arteca 1994) did not excrete any. Consequently,establishing cell lines that have outstanding secretion ability isrequired.

Cambium Culture

Cambium is a lateral meristem that is located on the lateral side of theplant. In the gymnosperm and woody dicotyledon plants, there is ahypertrophic growth due to the continual activity of the cambium; as aresult, giant plants having more than 11,000 years of the growth ringsexist. In genetics, meristems could be classified as primary andsecondary meristem. Primary meristem represents the meristem that formsduring embryogenesis and participates in the plant growth after seedgermination. Secondary meristem represents the meristem that is formedby dedifferentiation of the plant permanent tissue. Cambium is a primarymeristem with meristematic continuity derived from procambium withoutthe intervene of the permanent tissue.

Growth of this primary meristem is indeterminate and could be continuedif the conditions are given. Therefore, cambium culture has been usedfor rapid mass propagation of the cells.

In the preceding studies, cambium explants were prepared as follows:after the bark was peeled off, two longitudinal cuts, approximately 1 mmdeep in order to reach the xylem, were made into the woody stem at aninterval of 5 mm. The resulting explants were called ‘cambium’, whichwas constituted of part of the phloem, cambium and a small chip of xylem(Jouira et al., 1998).

It is reasonable to say that cells which are induced by the method asmentioned above are not the sole origin of cambium, but of multipletissues, which can be solemnly distinguished anatomically such asphloem, cambium and xylem. Thus, we could indicate that the methodmentioned above is not the ideal technique to separate only the cambiumelaborately from the various tissues that constitute the stems. Acreative method to separate only the cambium from the various tissues ofstems has been in demand.

The above information disclosed in this Background section is only forenhancement of understanding of the background of the invention andtherefore it may contain information that does not form the prior artthat is already known in this country to a person of ordinary skill inthe art.

SUMMARY OF THE DISCLOSURE

The present invention is provide to resolve the problems associated withthe prior art, e.g., variation occurred in plant cell culture.

One objective of the present invention is to provide a method ofproducing a single cell clone or a population of single cells byisolating and culturing only cambium from a plant.

Another objective is to provide a method of stably producingbiologically active substance (e.g., paclitaxel) or substances from thesingle cell clone or the population of single cells.

To achieve the above objectives, in one aspect, the present inventionprovides a method for isolating plant cambium-derived single cell clone,the method comprising: (a) preparing and then sterilizing a planttissue; (b) collecting the tissue containing cambium from the sterilizedplant tissue; (c) culturing the tissue containing cambium, and therebyinducing a cambium layer which is proliferated from cambium, and acallus layer which is derived from regions except cambium andproliferated in an irregular form; and (d) isolating the single cellclone from the cambium layer.

Preferably, the step (c) comprises culturing the said tissue in mediumcontaining auxin. In a preferred embodiment, the medium contains 1˜3mg/L of auxin.

In another aspect, the present invention provides a single cell cloneinduced from plant cambium, the single cell clone comprises thefollowing characteristics: (a) above 90% cells in suspension cultureexist as single cells; (b) having multiple vacuoles morphologically; (c)growing faster than the cell line derived from regions except cambium ofthe same plant origin, and culturing stably for a long time; (d) havinglow sensitivity to shear stress in the bioreactor; and (e) beinginnately undifferentiated.

Preferably, the plant is the genus Taxus. In a preferred embodiment, thegenus Taxus cambium-derived single cell clone has an ability ofreleasing paclitaxel 270-720 times more than the cell lines derived fromregions except cambium of the same plant origin.

In still another aspect, the present invention provides a method forproducing plant-derived biologically active substances, the methodcomprising the steps of: (a) producing the active substances byculturing the above single cell clone; and (b) collecting the activesubstances. Preferably, the culturing in the step (a) comprisesretrieving the media used in culturing of the single cell clone cultureand then supplying with a new media.

In a preferred embodiment, the single cell clone is the genus Taxuscambium-derived single cell clone, and the compound is paclitaxel. Inthis case, the media may further contain one or more materials selectedfrom the group consisting of methyl jasmonate, phenylalanine andchitosan.

In a further aspect, the present invention provides a method forpreserving a plant cell line. The method comprises cryoperservatingsingle cells derived from plant cambium, which are isolated by theabove-described method.

In accordance with the present invention, it is possible to culturesingle cell clone that has the meristematic continuity of primarymeristem without going through dedifferentiation by precisely separatingonly cambium from various tissues of woody plant twig or stem. Cell lineof the present invention allows stable production of biologically activesubstances due to less change in the cell growth rate and growth patternduring the long term culture. It is also optimal for the mass productionin commercial level because it is less sensitive to shear in abioreactor compared to the cell lines derived by the precedingtechniques, due to less aggregation and multiple vacuoles.

Metabolite activation can be stimulated by supplementing conditioningfactors to this cell line and cell vitality and biosynthesis can beextended as the cells releasing considerable amount of production intothe extracellular media through perfusion culture. High recovering rateafter cryopreservation due to homogeneity and division ability of thiscell line devises the establishment of cell bank. Through the presentinvention, close relationship between homogeneity of the cultures andvariation of secondary metabolites are confirmed, and the strategy forcommercialization may be developed as the variability of diversebiologically active substance production can be controlled and reduced.

The above and other features and advantages of the present inventionwill be apparent from or are set forth in more detail in theaccompanying drawings, which are incorporated in and form a part of thisspecification, and the following Detailed Description, which togetherserve to explain by way of example the principles of the presentinvention.

BRIEF DESCRIPTION OF DRAWINGS

The above and other features of the present invention will now bedescribed in detail with reference to certain exemplary embodimentsthereof illustrated the accompanying drawings which are givenhereinbelow by way of illustration only, and thus are not limitative ofthe present invention, and wherein:

FIG. 1A shows a stem after 30 days of cultivation in which cambium(represented by an arrow) is separated from callus cells derived fromtissue which consists of phloem, cortex, and epidermis; FIG. 1B shows asingle cell clone derived from cambium after 35 days of cultivation;FIG. 1C shows callus derived from tissue which consists of phloem,cortex, and epidermis after 40 days of cultivation; and FIG. 1D showscallus derived from embryo or needle after 50 days of cultivation.

FIG. 2 show growth rate expressed by total biomass production of threedifferent T. cuspidata cell cultures derived from cambium, embryo, andneedle in 6 months with the subculture interval ranged between 21 days.

FIG. 3 A-D show images of cell aggregation of two different T. cuspidatacultures derived from embryo or needle (A, C) and cambium (B, D), inwhich A shows large cell aggregates with the size of 1.5×10² μm, B showssingle cell population, D shows cells presenting a high density ofvacuole.

FIG. 4 is a graph showing the effect of elicitors and their combinationson paclitaxel production in T. cuspidata (single cell clone fromcambium) cultures, in which conditioning factors were incorporated tozero-day cultures; C, control; Chi, 50 mg/L Chitosan; Phe, 0.1 mMPheylanine; MJ, 100 μM Methyl Jasmonate; Com, combination of 50 mg/LChitosan, 0.1 mM Pheylanine, and 100 μM Methyl Jasmonate.

FIG. 5 is a graph showing the effect of conditioning factors onpaclitaxel production in T. cuspidata (single cell clone from cambium)suspension culture, in which elicitors were incorporated to 14-day oldcultures; CF, conditioning factors.

DETAILED DESCRIPTION OF THE DISCLOSURE

Practical examples of the invention are explained below. Induction andproliferation method of single cell clone from cambium is not onlyutilized in paclitaxel production system but it may also be utilized inall plant secondary metabolite production system. The following examplesare offered by way of illustration, not by way of limitation.

Practical Example 1 Preparation of Plant Materials and Isolation ofCambium

Seed, needle, twig of the yew tree were collected. After collecting thematerials, they were deposited in the solution of 100 mg/L ofantioxidant, ascorbic acid (L-ascorbic acid, DUCHEFA, The Netherlands)immediately and transferred and preserved. They were surface sterilizedby considering the morphology and physiological characteristics of thematerials.

1. Seed: After sterilizing the seeds with 70% ethanol for one minute,they were immersed in 1% Clorox solution for 48 hours and were washed 3to 4 times with sterile water. Next, embryo was separated from the seedin the solution of 0.5% PVP (polyvinyl pyrrolidone, DUCHEFA, TheNetherlands) and 50 mg/L of ascorbic acid (L-ascorbic acid, DUCHEFA, TheNetherlands), and 70 mg/L of citric acid (DUCHEFA, The Netherlands) andcultured on the callus induction media.

2. Needle and twig: After 24 hours of treatment with the solutioncontaining 1% Benomyl (Dongbu Hannong Chemical, Korea)+1% Daconil(Dongbu Hannong Chemical, Korea)+1% Streptomycin sulphate (DUCHEFA, TheNetherlands)+0.1% Cefotaxime sodium (DUCHEFA, The Netherlands), needlesand twigs were rinsed with tap water for 30 seconds to remove theremaining chemical substances and phenolic compounds. After sterilizingthem with 70% ethanol (DC Chemical, Korea) for one minute, 30% hydrogenperoxide (LG Chemical, Korea) for 15 minutes, 1% CLOROX solution for 15minutes, 3% CLOROX solution for 5 minutes in order, they were washed 3to 4 times with distilled water. To prevent the oxidation, both ends ofthe needle were cut in the solution of 5% PVP, 50 mg/L ascorbic acid and70 mg/L citric acid and cultured on the callus induction media.

3. Cambium preparation from the twig or stem: By holding the xylem whichis the center region of the twig or stem with the tweezers, phloem andcortex and epidermis tissues including cambium were peeled off. Thesepeeled tissues that contained cambium were laid on the media; cambiumwas allowed to touch the surface of the media.

Practical Example 2 Induction of Single Cell Clone from the IsolatedCambium

After 4th to 7th day of the culture, cell division of cambium wasobserved and on the 15th day of the culture, callus was beginning toform from the layer consisted of the phloem and cortex and epidermisthat were the upper part of the cambium. On the 30th day of the culture,the cambium began to be separated from the upper layer tissue thatcontained the phloem and cortex and epidermis; after these two layerswere completely separated naturally, they were cultured individually ondifferent petri dishes (FIG. 1).

For the purpose of cell and callus induction, universally known media ofthe plant cell and tissue culture could be used: e.g. mB5 (modifiedGamberg's B5 medium), MS (Murashige & Skoog medium), WPM (Lloyed &McCown), SM (schenk & Hildebrand medium), LP (Quoirin & Lepiovre).Application of all these media is possible. Various additives could besupplemented and components of the media could be reduced or eliminatedas the need arises. Among them, the most appropriate media was mB5. Thecontents of mB5 are described in the following Table 1.

TABLE 1 CELL LINE INDUCTION & MAINTENANCE MEDIUM IN TAXUS SPP.Composition Contents (mg/L) Inorganic salts KNO₃ 2500 (NH₄)₂SO₄ 134MgSO₄•7H₂O 121.56 MnSO₄•4H₂O 10 ZnSO₄•7H₂O 2 CuSO₄•5H₂O 0.025 CaCl₂•2H₂O113.23 KI 0.75 CoCl₂•6H₂O 0.025 NaH₂PO₄•H₂O 130.44 H₃BO₃ 3 Na₂MoO•2H₂O0.25 FeNaEDTA 36.7 Vitamin Myo-inositol 200 Thiamine-HCl 20 Nicotinicacid 2 Pyridoxine-HCl 2 L-ascorbic acid 50 Citric acid 75 Amino acidL-aspartic add 133 L-arginine 175 Glycine 75 Proline 115 Hormonea-Naphtalene acetic acid 2 Sucrose 10,000 Activated charcoal 100 Gelrite2,000

The cultures were grown on the media that was supplemented with a plantgrowth regulator, auxin (1-3 mg/L) in the dark at 25±1° C.

The cambium was composed of homogeneous cells, so its cell division wasuniform and proliferation occurred in the form of a plate. On the otherhand, the tissue containing the phloem and cortex and epidermisproliferated in irregular form because there was a discrepancy of celldivision due to the composition of many kinds of cells. There was aself-split of the layer in between the cambium and the tissue containingphloem and cortex and epidermis (FIG. 1). The cambium was homogeneousand the tissue containing phloem and cortex and epidermis washeterogeneous, so the self-split of the layer seemed to be the result ofdifferent division rate.

After 15th day of the culture, calli were formed on the explants ofembryo and needle that are composed of heterogeneous cells bydifferentiation and these calli proliferated in irregular forms due tothe different division rate of various cells just like the tissue thatcontained phloem and cortex and epidermis. (FIG. 1)

Practical Example 3 Establishment of Long Term Culture

Among the calli, white and friable calli that had good growth rate weresubcultured onto the new media every 21 days. The growth rate of theembryo and needle-derived cultures was very unstable and it often showedthe tendency of browning. On the contrary, the growth rate of thecambium-derived cultures was fast and there was no color change of thecultures. Therefore, it was possible to select the stable cells.

After six months of the culture, most of the embryo and needle-derivedcultures had yellow or light brown color and aggregation formed. Thecambium-derived cultures had white-yellow color and were maintained assingle cells or small cell clusters. The growth rate of the culturesthat turned brown and formed aggregation slowed down and the culturesdied eventually because of the phenol chemical substance that theyexcreted.

According to this inventor, maintenance and mass proliferation of theembryo and needle-derived cultures was difficult after 6 months, but thecambium-derived cultures were maintained stably for more than 20 monthsof the long term culture without any variation in the rate of cellgrowth, growth pattern and aggregation level (FIG. 2). In other words,variability appeared in growth pattern, depending on the homogeneity andheterogeneity of the initial plant materials.

Practical Example 4 Establishment of Cell Suspension Culture

The embryo and needle-derived and the cambium-derived cultures werecultured individually in the flask containing the liquid media (Table2).

TABLE 2 SUSPENSION MEDIUM IN TAXUS SPP. Composition Contents (mg/L)Inorganic salts Ca(NO₃)₂ 471.26 NH₄NO₃ 400 MgSO₄•7H₂O 180.54 MnSO₄•4H₂O22.3 ZnSO₄•7H₂O 8.6 CuSO₄•5H₂O 0.25 CaCl₂•2H₂O 72.5 K₃SO₄ 990Na₂MoO₄•2H₂O 0.25 H₃BO₃ 6.2 KH₂PO₄ 170 FeNaEDTA 36.7 VitaminMyo-inositol 200 Thiamine-HCl 20 Nicotinic acid 2 Pyridoxine-HCl 2L-ascorbic acid 50 Citric acid 75 Amino acid L-aspartic acid 133L-arginine 175 Glycine 75 Proline 115 Hormone a-Naphtalene acetic acid 2Sucrose 30,000

They were cultured on the 100 rpm rotating shaker in the dark at 25±1°C. With the two weeks of subculture interval, cultures were allowed tomaintain high vitality continuously as exponential growth phase.

Aggregation level which is the main cause of the variation of cellproductivity was measured. Cell aggregate quantification was measuredwith the biological microscope (CX31, Olympus, Japan). The result of theexperiment described above is on Table 3.

TABLE 3 TYPE OF CELL AGGREGATES OF TAXUS LONG-TERM CULTURES Large cellModerate Small cell Single cell Explant aggregates cell aggregatesaggregates morulation source 60% 30% 7%  3% embryo, needle  0  0 9% 91%cambium Large cell aggregates, size higher than 1.5 × 10² μm; Moderatecell aggregates, 1 × 10³ μm; Small cell aggregates, 4 × 10² μm < size <1 × × 10³ μm

In case of the suspension of the embryo and needle-derived cultures,about 60% had cell aggregation size more than 1.5 mm but in thesuspension of the cambium-derived cultures, 90% of the cells werecultured as single cells.

Practical Example 5 Scale Up

The embryo and needle-derived and the cambium-derived cultures werecultured in 3 L airlift bioreactor (Sung-Won SciTech, Korea) in the darkat 25±1° C.

In case of the embryo and needle-derived cultures, there was a greatvariability in the size and shape of the cells compared to the flaskculture. Diameter of the cell aggregation was enlarged up to 2-3 mm,which inhibited the flow inside of the bioreactor and developed unmixedregion in the bioreactor. Growth ring formed by the cells adhering tothe internal wall of the bioreactor. Cells in the center of the growthring died after 20 days because the media was not supplied efficiently.Eventually dead cells excreted toxic substances and these substanceslowered the vitality of all cells in the bioreactor. On the opposite,less aggregation of the cambium-derived cultures caused smooth aircirculation in the bioreactor; hence it was possible to diminish theamount of air supply from 200 ml to 150 ml per minute and the amount ofdeveloped bubble on the surface of the media was greatly reduced.

Doubling time of the embryo and needle-derived cultures in the flask was12 days but it was lengthened to 21 days in the bioreactor. It wasbecause of the growth ring formation and rapid decrease of cellviability due to sensitiveness to shear by cell aggregation and rigidcell wall. Doubling time of the cambium-derived cultures was 4 to 5 daysand there was no difference in the flask and the bioreactor, rather itwas shortened in the bioreactor (Table 4). The cambium-derived culturesformed very small growth ring in the bioreactor and the growth ring wasdissolved easily by agitating the media with a simple stimulus.Moreover, there was no decrease in cell viability due to lesssensitivity to shear by less cell aggregation and multiple vacuoles.

TABLE 4 RELATIONSHIP BETWEEN DOUBLING TIME PATTERNS AND EXPLANT SOURCEIN T. CUSPIDATA CELL CULTURES IN FLASK AND BIOREACTOR Doubling time(day) Explant source flask bioreactor embryo 11.5 21 needle 12 21cambium 5 4

Practical Example 6 Elicitor

Elicitor controls molecular signal in plant cells and is widely used forthe increase of secondary metabolite productivity. After the treatmentof methyl jasmonate as an elicitor and 10 other kinds of elicitors, weobserved that methyl jasmonate had positive effect on the paclitaxelproduction. It was possible to obtain relatively high metabolitesproductivity through the combination of methyl jasmonate and otherelicitors. Especially, paclitaxel production was very effective with thetreatments of methyl jasmonate, chitosan and phenylanine (FIG. 4).

Practical Example 7 Conditioning Factors

Plant derived secondary metabolites are produced when the cells aregrowing or when the cells stopped growing. Therefore, two stage culturesare suitable for the production of metabolites like paclitaxel whosecell growth stage and metabolite production stage are separated. In thefirst stage, cells were proliferated in a large scale by optimizing thecell growth and in the second stage, the culture condition was changedfor the optimization of metabolites production.

Cell lines with high secondary metabolites productivity grow slower anddie faster than the cell lines with low productivity. Therefore, massproliferation is difficult and mass production of the metabolites isimpossible.

In this invention, cell lines with the ability of low proliferation andhigh production were not used for the proliferation in large scale,rather they were used as the helper cells that have the conditioningfactors for the production of secondary metabolites. We observed thepaclitaxel production after adding the helper cells. The results aresummarized in FIG. 5.

Practical Example 8 Perfusion Culture

On the 14th day of culture, elicitor was treated to the embryo andneedle-derived and the cambium-derived cultures. From the point ofelicitation, spent media was retrieved in an aseptic condition withpipette on every 5 days and was supplied with the same amount of newmedia simultaneously. The production of paclitaxel in the cell and themedia were observed after 45 days of the long term culture. The resultwas summarized in Table 5.

TABLE 5 PACLITAXEL PRODUCTION AND RELEASE OF T. CUSPIDATA CELLS INVARIOUS EXPLANT SOURCES AND PROCESSES Materials & Taxol yield (mg/kg)Taxol release processes In cell In medium Total (days) (%) embryo 12.970.03  13 (28) 0.2 needle 10.92 0.08  11 (28) 0.7 cambium 76.4 21.6  96(28) 22 cambium 0 0  0 (45) — cambium 69 195 264 (45) 74 perfusionculture Medium renewal incorporated into the cell cultures 5 days afterelicitation, which was carried out by adding 50 mg/L chitosan, 0.1 mMpenylanine and 100 μM methyl jasmonate to 14-day-old cultures. Theexperiment with repeated medium renewal every 5 days.

Depending on the cell lines, paclitaxel release of the cell to the mediawas different. Paclitaxel releasing ability of the cambium-derivedcultures was superior to the cultures of the preceding techniques. Thecambium-derived single cell clone had an ability of releasing paclitaxelabout 270˜720 times more than the cell lines derived from embryo andneedle (in medium of Table 5). Moreover, application of perfusionculture facilitated the release of secondary metabolites to the media.Improvement in the extracellular release of secondary metabolitesthrough the cambium-derived single cell clone by exchanging the mediaperiodically had great importance because it allowed continuous recycleof the biomass and simple purification.

In other words, periodical exchange of the media in the cambium-derivedsingle cell clone culture can be considered as a stable method ofproducing valuable metabolites in the long term culture, because itprevents feedback inhibition of accumulated metabolites in the cell,degradation and conversion of the metabolites in the media.

Practical Example 9 Cryopreservation

On the 6th or 7th day of the culture, suspension cells were pre-culturedin the media containing 0.16M of manitol for 3 days at the roomtemperature and then maintained at 4° C. for 3 hours. Cells wereharvested and placed into 4 ml cryovial which had the media containing40% ethylene glycol (Sigma, USA) and 30% sorbitol (DUCHEFA, TheNetherlands) and cultured for 3 minutes at 4° C.

Suspension cells that were treated with cryopreservatives were frozenafter the cells were soaked in the liquid nitrogen. For thawing,cultured cells in the liquid nitrogen for more than 10 minutes werethawed in the 40° C. water bath for 1-2 minutes. For the re-growth ofthe cells, cryopreserved cells were transferred onto the semi-solidgrowth media (Table 1) containing 0.5 M sorbitol and alleviated at theroom temperature for 30 minutes. Cells were cultured on the semi-solidgrowth media containing 0.1M sorbitol for 24 hours. And then, the cellswere cultured on the semi-solid growth media without sorbitol for 24hours, twice. Cell viability was evaluated.

Practical Example 10 Analysis of Paclitaxel Content

After separating the cells from the media of the recovered samples,paclitaxel contents were analyzed. Cell mass was measured after dryingthe cells completely with vacuum desicator (Sam Shin Glass, Korea).About 100 mg (dry weight) of the cells were mixed with 4 ml solution(1:1 v/v) of methanol (Sigma, USA) and methylchloride (Sigma, USA) andwere extracted by ultrasonic cleaner (Branson, USA) for 3 times in onehour interval at the room temperature. Cells were fully dried andextracted several times by using 4 ml of methylchloride. Separatedorganic solvent layer was vacuum dried and the remaining was dissolvedin 1 ml of methanol. Dissolved extract was agitated equally byultrasonic cleaner. Then, after centrifugation, the pellet was removed(8,000 g×5 min).

Media (1-5 ml) that was separated from the cell was combined with thesame volume of methylchloride and was extracted 3 times after fullagitations. After organic solvent was vacuumed and dried completely, itwas dissolved in 0.5 ml of methanol again.

HPLC (High Performance Liquid Chromatography, Shiseido, Japan) was usedfor the analysis of the content and Sigma products were used forpaclitaxel standard substances. Capcell pak (C18, MGII, 5 um, 3.0mm.times.250 mm, Shiseido, Japan) was maintained to 40° C. by using theoven, and water and acetonitril (Burdick & Jackson, USA) (50:50, v/v)were combined for the mobile phase and dropped regularly with the speedof 0.5 ml/min. UV-VIS detector (227 nm, Shiseido, Japan) was used.

As described above, according to the present invention, single cellclone, a primary meristem which has the meristematic continuity withoutdedifferentiation, obtained by separating cambium purely from twig orstem resulted in higher productivity due to shorter doubling time and/ordue to less change in the cell growth and growth pattern during the longterm culture than the cell lines obtained by conventional techniques. Inaddition, the-thus obtained single cell clone facilitated scale upbecause of less aggregation and multiple vacuoles of the cell lines. Thesingle cell clone also allowed recovery after cryopreservation withoutany genetic variation.

1. A method for preparing an isolated population of cells, the methodcomprising: (a) providing a tissue from a plant; (b) obtaining from theplant tissue a tissue containing cambium; (c) culturing in a medium thetissue containing cambium; and (d) isolating the population of cellsfrom the cambium, wherein the isolated cells are innatelyundifferentiated.
 2. The method according to claim 2, wherein in thestep (c), the medium includes auxin.
 3. The method according to claim 2,wherein the medium contains 1-3 mg/L of the auxin.
 4. The methodaccording to claim 1, wherein the plant is the genus Taxus.
 5. Themethod according to claim 1, further comprising sterilizing the planttissue.
 6. The method according to claim 1, wherein in the step (c), thecambium is induced to proliferate cells and in the step (d), thepopulation of cells is isolated from the cells proliferated from thecambium.
 7. The method according to claim 6, wherein in the step (c),the medium includes auxin.
 8. The method according to claim 7, whereinthe medium contains 1-3 mg/L of the auxin.
 9. The method according toclaim 6, wherein the plant is the genus Taxus.
 10. The method accordingto claim 6, further comprising sterilizing the plant tissue.
 11. Amethod for producing a biologically active substance or substances, themethod comprising the steps of: (a) producing the active substance orsubstances by culturing in a medium the isolated population of cellsprepared by the method of claim 1; and (b) collecting said activesubstance or substances.
 12. The method according to claim 11, whereinin the step (a), a predetermined amount of the medium that has been usedto culture the cells is removed and a predetermined amount of a newmedium is introduced.
 13. The method according to claim 11, wherein theplant is the genus Taxus and the active substance is paclitaxel.
 14. Themethod according to claim 13, wherein the medium includes at least oneselected from the group consisting of methyl jasmonate, phenylalanine,and chitosan.
 15. A method for preserving the isolated population ofcells prepared by the method of claim 1, which comprises cryopreservingthe isolated population of cells.